Despite the strong relationship of elevated plasma levels of high density lipoproteins (HDL) with protection from cardiovascular disease (CVD), the molecular details of how newly secreted lipid-free apolipoproteins transition to lipidated HDL particles remains unknown. This gap in understanding is a critical hindrance as the pharmaceutical industry is in the midst of a major effort to identify therapies that raise plasma HDL levels. Our long term goal is to understand how HDL is generated and whether this process can be manipulated for therapeutic benefit. Pursuant to this, our objective here is to derive a molecular understanding of the lipid binding structural transitions of two prototypical HDL apolipoproteins: apolipoprotein (apo)A-I and apoA-IV. Since these proteins interact with lipid in distinct ways, we aim to define a range of mechanistic themes that will be applicable to the broader apolipoprotein family. Our central hypothesis is that apolipoprotein self-association is a critical feature that dictates a given protein's ability to generate HDL. This is based on the fact that apolipoproteins universally self-associate as well as our preliminary data showing a clear and highly novel mechanism for how they accomplish this. Our rationale is that understanding apolipoprotein structural adaptations during lipid binding will provide new insights into the mechanism of HDL formation in vivo. To test our hypothesis, we will pursue three specific aims: 1) Determine the structure and self-association mechanism of lipid-free apoA-I and apoA-IV; 2) Determine the structure of apoA-I and apoA-IV in their lipid-bound states; and 3) Determine the importance of self-association and test molecular folding pathways for apoA-I and apoA- IV lipoprotein particle assembly. In Aims 1 and 2 we will derive badly needed high-resolution structures of these apolipoproteins at the beginning and end of the HDL particle assembly process by X-ray crystallography, complemented by solution-based spectroscopic and cross-linking experiments. This knowledge will be used in Aim 3 to alter apoA-I and apoA-IV self-association properties and test the effects on ability to generate HDL particles either by spontaneous lipid reorganization or via cell-based HDL particle formation assays. We will also introduce disulfide constraints to test detailed schemes for the lipid-binding structural transitions undertaken by both proteins. This work is innovative because we have discovered a novel helix swapping mechanism that explains apoA-IV self-association and likely other apolipoproteins as well. This provides a new conceptual framework upon which a better understanding of apolipoprotein function can be built and it offers a clear basis for the arrangements that apolipoproteins adopt in HDL particles. Finally, the work is significant because it will illuminate the molecular events that occur during HDL formation as well as mechanisms for dissociation of apolipoproteins from pre-formed HDL particles, both critical processes governing HDL metabolism. This understanding will help guide the development of therapeutic strategies designed to raise plasma HDL for protection against CVD, the number 1 killer in the U.S.